Bartonella bacilliformis is the etiologic agent of Oroya fever in humans. The bacterium selectively parasitizes erythrocytes, culminating in one of the most severe hemolytic anemias known. The long-term objective for this research is to use B. bacilliformis as a model for investigating the pathogenesis of bacteria which engage in hemotrophic parasitism of erythrocytes. Hemotrophy is a survival strategy employed by a number of bacterial pathogens. Data suggest that three factors are involved in the virulence of B. bacilliformis, including an adhesin (erythrocyte binding), the flagella (host cell entry), and a beta-hemolysin protein (erythrocyte entry and/or exit). The overall goal of this project is to molecularly characterize these putative virulence factors of B. bacilliformis and to assess their importance to pathogenesis. To this end we will clone and sequence the gene(s) for each determinant. Cloning strategies include the use of degenerate oligonucleotides to map and clone genes from our previously constructed lambda genomic DNA library of B. bacilliformis, shotgun cloning, and finally by insertional inactivation of expression by transposon (Tn) mutagenesis and subsequent localization and identification of the interrupted gene(s) by DNA hybridization. DNA recombinants containing the gene of interest will be identified by functional gene expression, DNA hybridization, or by detection of expressed protein using antiserum generated against each determinant. DNA sequence analysis of each determinant gene will provide a means of predicting primary and secondary protein structure and will allow us to compare the gene or protein to database homologues from other bacteria. To assess the importance of each virulence factor to pathogenesis, monoclonal antibody and Tn mutagenesis will be directed against the protein and gene of each determinant, respectively. Tn mutants will be analyzed by DNA sequencing to verify that determinant genes are insertionally inactivated. Tn mutants and wild-type bacteria treated with neutralizing antibody will be used to determine if infectivity is reduced in vitro by assays focusing on impaired erythrocyte adhesion, invasion, and inability to escape from erythrocyte endosomal vacuoles. Data from this study will greatly increase our understanding of molecular aspects of pathogenesis in B. bacilliformis, and hemotrophic bacteria in general.